Efficient delivery of a nucleic acid to a desired target site has been the focus of many intense studies. Once introduced to the target site, the nucleic acid, may exert, directly or indirectly, a biological effect in the target site. In some instances, the delivery of the nucleic acid may take use of carriers that are designed to deliver the nucleic acid to the target site. Exemplary nucleic acids that may be delivered to a target site include deoxyribonucleotides nucleic acid (DNA) and ribonucleotides nucleic acids (RNA), such as, for example, siRNA, miRNA, shRNA, Antisense RNA (AS-RNA), and the like.
RNA interference (RNAi) is an endogenous cellular mechanism of gene silencing. RNAi is carried out by double-stranded RNA (dsRNA) that suppress the expression of specific genes with complementary nucleotide sequences either by degrading specific messenger RNA (mRNA) or by blocking mRNA translation. RNAi can be also activated exogenously by expressing short hairpin RNA (shRNA) with viral vectors, or by incorporating synthetic small interfering RNA (siRNA) directly into the cell cytoplasm. siRNAs are chemically synthesized double stranded RNAs (dsRNAs) of 19-23 base pairs with 2-nucleotides unpaired in the 5′-phosphorylated ends and unphosphorylated 3′-ends. Inside the cell cytoplasm, siRNAs are incorporated into RNA induced silencing complex (RISC), a complex that separates the strands of the RNA duplex and discards the sense strand. The antisense RNA strand then guides RISC to anneal and cleave the target mRNA or block its translation. By recycling the target mRNA, the RISC complex incorporating the anti-sense strand may show a therapeutic effect for up to days in dividing cells and for several weeks in non-dividing cells. Furthermore, repeated administration of siRNAs can result in stable silencing of its target. However, despite this promise, utilizing siRNAs as therapeutics is not a trivial task. For example, due to the large molecular weight (˜13 kDa) and the net negative charge, the efficiency with which naked siRNAs molecules cross the plasma membrane and enter the cell cytoplasm is usually very low. When injected intravenously, in addition to rapid renal clearance and susceptibility to degradation by RNAses, unmodified naked siRNAs are recognized by Toll-like receptors (TLRs). This often stimulates the immune system and provokes interferon response, complement activation, cytokine induction, and coagulation cascades (Reviewed by Peer D.). Beside the undesired immune activation, those effects can suppress gene expression globally, generating off-target and misinterpreted outcomes.
For in-vitro or ex-vivo delivery of siRNA to cells, conventional transfection methods are generally used (reviewed by Weinstein and Peer). In-vivo delivery of siRNA can be classified into two groups: localized or systemic. Local delivery of siRNAs has been demonstrated in various animal models and is employed in several ongoing clinical trials. Based on local injections of naked or cationic lipid/polymer-formulated siRNAs, this method of delivery is mainly suitable for mucosal diseases, subcutaneous tissues, intraocular injections to the vitreous body of the eye, and the like. Systemic delivery of siRNAs provides additional complications. Whereas cellular and local delivery deal with the need for internalization, release, and accumulation of the siRNAs in the cell cytoplasm, systemic delivery in an entire animal enforces additional hurdles such as, for example, the siRNAs interaction with blood components (which is a common complication using cationic liposomes due to the electrostatic interaction of the positive charge of the liposome with the generally negative charge of serum proteins), entrapment within capillaries, uptake by the reticuloendothelial cells, degradation by RNAses, anatomical barriers (such as the liver, spleen and filtration by the kidneys), immune stimulation, extravasation from blood vessels to target tissues, permeation within the tissue, and the like.
Various methods and carriers have been suggested over the years for systemic delivery of siRNA molecules. The methods and carriers include passive delivery of the siRNA or targeted delivery of the siRNA. Exemplary carriers described in the art include: Stable nucleic acid-lipid particles (SNALP), neutral liposomes, lipidoid containing liposomes, atelocollagen, cholesterol-siRNA, dynamic polyconjugates, PEI nanoplexs, antibody-protamine fusion proteins, aptamer-siRNAs, targeted cationinc liposomes and cyclodextrin containing polycation (CDP) (reviewed by Manjunath and Dykxhoorn; and Weinstein and Peer.)
Some of the siRNA carriers described in the art make use of hyaluronic acid that may be used as component of the particle and/or as a targeting moiety. For example: A publication by Taetz et. al., is directed to Hyaluronic acid modified DOTAP/DOPE liposomes for the targeted delivery of antitelomerase siRNA to CD44 Expressing Lung cancer cells. A publication by Lee. et. al. is directed to target specific intracellular delivery of siRNA using degradable hyaluronic acid nanogels. A publication by Choi et. al., is directed to self assembled hyaluronic acid nanoparticles for active tumor targeting. A publication by Peer et. al., is directed to Systemic Leukocyte-Directed siRNA Delivery Revealing Cyclin D1 as an Anti-Inflammatory Target. For example, PCT patent application publication no. WO 2011/013130 is directed to cell targeting nanoparticles comprising polynucleotide agents and uses thereof. Additionally, U.S. Pat. No. 7,544,374 is directed to lipidated glycosaminoglycan particles and their use in drug and gene delivery for diagnosis and therapy.
Nevertheless, the carriers described in the art, including carriers making use of hyaluronic acid do not address all the hurdles associated with a successful delivery of siRNA to its target cell, and in particular, in-vivo delivery.
There is thus a need in the art for improved carrier compositions for the efficient and specific delivery of siRNA into a desired target site, wherein the carrier compositions are stable, have a long shelf life, biodegradable, amenable to industrial production processes, have high encapsulating capacity, non toxic, avoid induction of immune responses, provide enhanced protection (stability and integrity) to the siRNA encapsulated therein and are able to efficiently deliver in-vitro and in-vivo, the siRNA to its target site, such that the siRNA is able to efficiently exert a desired effect.